Conjugates of low molecular weight haptens and a synthetic copolymer of D-glutamic acid and D-lysine (D-GL) have been shown to be very effective in inducing in experimental animals hapten-specific immunological tolerance which is highly specific and long-lasting (for review see Katz, 1974; Katz and Benacerraf, 1974). The tolerant state in such circumstances is (1) restricted to bone marrow-derived lymphocytes (B cells) which are precursors of antibody-secreting cells, (2) accompanied by a significant diminution of hapten-specific antigen-binding B cells, and (3) results in a preferential depression of the high affinity anti-hapten antibody response. Antibody responses of all immunoglobulin classes, including reaginic (IgE) anibodies responsible for local and systemic allergic reactions, are abolished by hapten-D-GL conjugates. Moreover, a very important aspect of this system is that such D-GL conjugates are highly effective in turning off ongoing antibody responses in previously sensitized individuals.
This system has been well-characterized with 2,4-dinitrophenyl (DNP)-D-GL (Katz, 1974; Katz and Benacerraf, 1974) and has been extended to the induction of tolerance to nucleoside conjugates of D-GL (Eshhar et al., 1975) which has clinical potential for abolishing anti-nuclear antibody production occurring in patients with systemic lupus erythematosus. Induction of tolerance to the major allergenic determinant of penicillin, the benzylpenicilloyl (BPO) hapten, has also been demonstrated by administering BPO-D-GL to experimental animals (Chiorazzi et al., 1976); the latter system has obvious clinical applicability in terms of treating patients with penicillin allergy. Based on the previously established knowledge in tolerance systems using hapten-D-GL conjugates, it is conceivable that larger macromolecules coupled to D-GL will have similar tolerance-inducing properties, once bound to specific immunoglobulin receptors on B lymphocytes. Therefore, I have been attempting to develop the methadology for preparation of stable conjugates of complex proteins coupled to D-GL for therapeutic use.
There are two major concerns in the preparation of protein-D-GL conjugates which will be tested in experimental animals for their biological activities and clinical applicabilities. (1) The conjugation reaction should be as mild as possible so that the antigenic determinants of the protein of interest are maximally retained. (2) The conjugate should be free of non-conjugated protein, especially protein dimers or oligomers which may be produced under the conjugation conditions and may not be easily separable from the conjugate by conventional chromatographic techniques. For all the protein-D-GL conjugates prepared, there should be a method to demonstrate conclusively the absence of non-conjugated protein, since contamination of any preparation by such molecules would pose a serious detriment to the effectiveness of tolerance induction and, more importantly, could constitute a life-threatening health hazard if such preparations were employed clinically.
Most commonly used coupling reagents such as glutaraldehyde, bisimidoesters, toluenediisocyanate and carbodiimides are not suitable for our purpose since they react mainly by coupling amino group with amino or carboxyl group and can result in extensive self-coupling of proteins. D-GL, which has an abundance of amino and carboxyl groups is particularly susceptible to this process. The ideal coupling method involves the introduction of a functional group into the protein (or D-GL) which reacts only with another functional group introduced into D-GL (or the protein). The recently reported coupling reagent m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), Kitagawa and Aikawa, 1976) seems to be just such a reagent. Sulfhydryl groups, the other necessary reactive component, can be incorporated into D-GL (or protein) by known procedures.
Protein conjugates with D-GL are prepared by, first, preparing MBS modified protein and SHA modified D-GL and reacting MBS modified protein with SHA modified D-GL to produce protein-D-GL conjugates, and, conversely, by preparing SHA modified protein and MBA modified D-GL and reacting SHA modified protein and MBA modified D-GL to form the same class of protein-D-GL conjugates. In a preferred embodiment of the invention, MB-Antigen and SH-D-GL are reacted to form D-GL-Antigen conjugate, or conversely, SH-Antigen and MB-D-GL are reacted to form the same class of D-GL-Antigen conjugate. Specific novel methods for carrying out the preparation of MB-protein, SH-protein, MB-D-GL and Sh-D-GL and reacting MB-protein with SH-D-GL or SH-protein with MB-D-GL to form the D-GL-protein conjugate are specific features of the invention. More particular, individual exemplary features of the invention are the preparation of MBS modified antigens such as MB-In, and MB-E, SHA modified antigens such as SH-In and SH-E, and MB-D-GL or SH-D-GL followed by the reaction of one of the MB-Antigens with SH-D-GL or one of the SH-Antigens with MB-D-GL to prepare D-GL-Antigen, e.g., D-GL-E or D-GL-In conjugates of, respectively, Ragweed antigen E or insulin.
Preparation and isolation of protein-D-GL conjugates, demonstrated by the exemplary OVA-D-GL conjugates, by the generation of SH-D-GL (biotin) from its protected precurser in situ in the presence of MB-Protein, and the purification by direct application of the reaction mixture to an avidin-Sepharose column are additional features of the present invention.
Other features of the invention include the specific reaction conditions, preparation and separation techniques and methods described in detail hereinafter.
It is pointed out that the OVA-D-GL conjugate system, the starting and intermediate reagents, and the reactions using ovalbumin, and the information related to these reactions, materials and procedures, are set forth in detail to exemplify the invention and not as a statement or implication of the scope of the invention. The invention is a general method for forming protein-D-GL conjugates. A particular application of the invention is the preparation of D-GL-Antigen conjugates. Ovalbumin reactions typify protein reactions and, therefore, are selected as illustrative only of a general invention.